This invention relates to a process for the preparation of molecularly imprinted polymers useful for separation of enzymes. Molecularly imprinted polymers prepared by the process of the invention exhibit selective binding of imprinted enzyme, which is useful in separating the imprinted enzyme from aqueous solution of the imprinted enzyme or a mixture containing imprinted enzyme and other enzymes.
Purifications and isolations of biological macromolecules like proteins and enzymes are critical multi-stop processes as they result in very low yields and thus high cost. As a better alternative to the conventional processes, researchers have developed various techniques for purification of enzymes which can broadly be classified as follows.
1. Affinity chromatography (Y. Li, G. Kunyu, C. Lubai, Z. Hanfa, Z. Yunkui Sepu, 14:415 (1996), T. Makriyannis, Y. D. Clonis, Biotech. Bioengg. 53.49 (1997)).
2. Affinity precipitation (C. Senstad, B. Mattiasson, Biotech. Bioengg. 33:216 (1989), M. Schneider, C. Guillot, B. Lamy, Ann. N.Y. Acad. Sci. 369:257 (1981)).
3. Affinity crossflow ultrafiltration (K. Sigmundsson, H. Filippusson, Polymer int. 41:355(1996), T. B. Choe, P. Masse, A. Verdier, Biotech. Lett. 8:163 (1986)).
4. Affinity partitioning (G. Takerkart, E. Segard, M. Monsigny, FEBS Lett. 42:218 (1972), B. A. Andrews, D. M. Head, P. Dunthorne, J. A. Asenjo, Biotech. Tech. 4:49 (1990)).
5. Immobilized metal affinity chromatography (G. Ehteshami, J. Porath, R. Guzmnan, G. Ehteshami, J. Mol. Recognit. 9:733 (1996), A. L. Blomkalns, M. R. Gomez, Prep. Biochem. Biotechnol. 27:219 (1997)).
6. Molecularly imprinted immobilized metal affinity chromatography (F. H. Arnold, P. Dhal, D. Shnek, S. Plunkett, U.S. Pat. No. 5,310,648 (1994)).
These techniques exploit either the binding interaction between active site of an enzyme and its inhibitor i.e. affinity monomer or between surface histidine, tyrosine, cysteine groups of enzymes with metal ions immobilized on a support. But these techniques also suffer from one or the other disadvantages as follows.
Affinity chromatography uses a column containing an inhibitor or a dye or an antibody for a given enzyme for its separation from a mixture of enzymes. The solution of enzymes is poured over the affinity column to retain the desired enzyme on column for subsequent isolation. This technique is efficient only for small capacity columns. With the scale up of columns, the problems of sample pretreatment and plugging of packed column become severe.
The affinity precipitation technique needs a complex to be formed between a macro-ligand and a protein both containing multiple binding sites. When such a complex is formed, it precipitates. Thus, this technique is restricted mainly for multimeric proteins. Beside such macro-ligands are often costly. Therefore, certain synthetic inhibitors or dyes are linked to stimuli responsive polymer which can be precipitated by pH or temperature stimuli after it forms a complex with the enzyme. In this case along with the desired enzyme, a substantial amount of other enzymes bind to the polymer by means of nonspecific adsorption.
In case of affinity crossflow ultrafiltration, a solution of enzymes is filtered through a membrane containing affinity group under pressure. This technique is suitable in cases where the difference between molecular weights of two enzymes is high. Also, with increase in filtration time, denaturation of enzyme as well as clogging of the membrane takes place due to pressure applied.
Affinity partitioning of two-phase aqueous systems is widely used technique as compared to those mentioned above. In this technique concentrated aqueous solution of poly (ethylene glycol) (PEG) with or without linking affinity group is mixed with enzyme solution containing moderate a high salt concentration. The two phases are mixed well and allowed to separate. The enzyme gets predominantly partitioned in one phase, which subsequently can be isolated. Disadvantages of this technique are nonspecific extraction of other proteinaceous molecules along with desired enzyme and also poor interactions between enzyme and affinity group due to high ionic strength.
Immobilized metal affinity chromatography is a technique in which columns of polymeric support containing chelated metal ions are used. These metal ions form coordination complex with histidine, tyrosine, crysteine etc present on the surface of the enzyme. Although this technique has advantages like high column capacity, ease in enzyme elution etc. it is not very selective in discriminating between two closely related (with respect to their amino acid sequences) enzymes.
Molecular imprinting of matrices containing metal chelates is a recently developed technique, which increases the selectivity (F. H. Arnold, P. Dhal, D. Shnek, S. Plunkett, U.S. Pat. No. 5,310,648 (1994)). In this technique, complex of monomer containing chelated metal ion and enzyme is polymerized with crosslinker in order to imprint the polymer with enzyme. Although this technique exhibits a substantial selectivity, it is not as selective as that of biological antibodies or active site inhibitors of enzymes.
Synthetic inhibitors are molecules which exhibit affinity to the active site of a given enzyme. This interaction is very specific for a given pair of enzyme and inhibitor and such inhibitors bind very strongly to a given enzyme.
Molecular imprinting is a simple technique which allows synthesis of polymers capable of recognizing specific molecules. These polymers are stable and can withstand harsh conditions such as temperature and pH etc (G. Wolff, Molecular interactions in Bioseparations Ed. by T. T. Nao., Plenum press, N.Y. (1993)). Thus, these polymers are finding wide range of applications. A polymer containing monomer based on enzyme inhibitor i.e. affinity monomer which is also imprinted for corresponding enzyme is expected to exhibit a high selectivity as well as high capacity for binding a specific enzyme. This synergistic affinity-imprinting effect will not be available in the above mentioned techniques. Such affinity-imprinted polymers have not been reported so far.
The object of the present invention is to provide a process for the preparation of molecularly imprinted polymers comprising affinity monomers imprinted for various enzymes, affinity imprinting technique, useful for achieving high selectivity as well as high capacity for separation of enzymes.
Accordingly the present invention provides a process for the preparation of molecularly imprinted polymers useful for separation of enzymes which comprises the steps of:
(i) reacting the complex of enzyme and affinity monomer, a comonomer and a crosslinker, with a polymerization initiator and a polymerization accelerator at ambient temperature and pressure for a period ranging between 2 to 24 hrs. thereby obtaining a crosslinked polymer,
(ii) crushing the cross linked polymer obtained to fine particles,
(iii) adding a solvent and extracting imprinted enzyme from the polymer,
(iv) obtaining the molecularly imprinted polymer, and
(v) contacting the imprinted polymer with aqueous solution containing imprinted enzyme or a mixture of imprinted enzyme and other enzymes and isolating the enzyme-adsorbed polymer.
In one embodiment, the enzymes are selected from the group of enzymes and proteins comprising trypsin, chymotrypsin, lysozyme, and ovalbumin (nonenzyme nutritional protein).
In another embodiment, the complexing affinity monomers are selected from the group comprising acryloyl derivatives of inhibitors for respective enzymes such as N-acryloyl para amino benzamidine, N-acryloyl 4-phenyl butyl amine, acryloyl N-acetyl glucosamine, and N-oleoylacrylamide respectively, and the enzyme-affinity monomer complex may be prepared by contacting aqueous solutions of enzyme and the respective affinity monomer.
In another embodiment, affinity monomers, N-acryloyl para amino benzamidine, N-acryloyl 4-phenyl butyl amine, acryloyl N-acetyl glucosamine and N-oleylecrylamide may be prepared by the reaction of acryloyl chloride with para amino benzamidine, 4-phenyl butyl amine, N-acetyl glucosamine and acrylamide respectively.
In yet another embodiment, the ratio of enzyme to affinity monomer may be in the range of 1:1 to 1:10, preferably 1:1 (w/w).
In a further another embodiment, the comonomer may be selected from compounds such as acrylamide, methacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, acrylic acid, methacrylic acid and the like.
In another embodiment, the amount of comonomer in the polymerization mixture may be in the range of 5 to 80%, more particularly 45 to 55% by weight of the total feed (i.e. enzyme-affinity monomer complex=comonomer-crosslinker, hereinafter).
In still another embodiment, crosslinker may be selected from compounds such as methylene bisacrylamide, N, N diacryloyl 1,2 diaminoethane, N, N dimethacryloyl 1,2 diaminoethane, N, N diacryloyl 1,6 diaminohexane, ethylene glycol dimethacrylate and the like.
In still another embodiment, the amount of crosslinker may be between 10% to 90%, more particularly 30 to 50% by weight of the total feed.
In yet another embodiment, the polymerization initiator used may be selected from compounds such as ammonium persulfate, ceric ammonium sulfate, potassium persulfate, azobisisobutyronitrile and the like.
In yet another embodiment, the amount of polymerization initiator may be 0.1% to 1% by weight of the total feed.
In still another embodiment, the polymerization accelerator may be selected from compounds such as ethylene diamine, N, N, N, N tetramethylene ethylenediamine and the like.
In still another embodiment, the amount of polymerization accelerator may be 1% to 4% by weight of the total feed.
In still another embodiment, the solvent used to extract the enzyme from the imprinted polymer may be selected from solvents which break the enzyme-inhibitor complex such as phenol, chloroform, glacial-acetic acid and the like.
In a feature of the present invention, the molecularly imprinted polymer is typically prepared under mild conditions as follows. An affinity monomer and its respective enzyme are dissolved in water and stirred for 15 to 30 minutes at room temperature to form the enzyme-affinity monomer complex. Then comonomer, crosslinker and polymerization initiator are added to the solution containing enzyme-affinity monomer complex and the solution is purged with nitrogen for 10 minutes. Polymerization accelerator is then added and the solution is kept at 37xc2x0 C. for 18 hours for polymerization. The crosslinked polymer so obtained is treated with acetone to remove water and crushed to small particles. Template enzyme is extracted from the particles by 3 to 4 alternative treatments of the solvent which breaks the complex between enzyme and the inhibitor. Polymer particles are then sieved and used for enzyme separations.
In another feature of the present invention, enzyme imprinted polymers are contacted with aqueous solution containing the respective enzyme or a mixture of enzymes and allowed to shake at room temperature for 1 hour. Polymer particles with bound enzyme are then filtered and isolated.
Although the present invention describes a process for the preparation of molecularly imprinted polymers useful for the separation of enzymes, namely trypsin, chymotrypsin, lysozyme and ovalbumin (nonenzyme nutritional protein), the scope of the present invention is not and should not be construed to limit only to such molecularly imprinted polymers for separation of trypsin, chymotrypsin, lysozyme and ovalbumin only but it may extend to such combinations of affinity monomers and their respective enzymes.
The ranges and limitations provided in the instant specification and claims are those which are believed to particularly point out and distinctly claim the present invention. It is however understood that other ranges and limitations which perform substantially the same function in the same or substantially the same manner to obtain the same or substantially the same results are intended to be within the scope of the instant invention as defined by the instant specification and claims.